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Results for “egg activation”

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PubMed1993

Complete mouse egg activation in the absence of sperm by stimulation of an exogenous G protein-coupled receptor.

Sperm-induced egg activation has several features in common with signal transduction pathways mediated by ligand-receptor-effector systems. G proteins are postulated to act as signal transducers regulating the plethora of cellular responses constituting egg activation that occur in response to sperm. We report that acetylcholine treatment of mouse eggs expressing the human m1 muscarinic receptor, a G protein-coupled receptor, results in the full complement of events of egg activation, including ZP2 to ZP2f conversion, recruitment of maternal mRNAs, pronuclear formation with subsequent DNA synthesis, cleavage to the two-cell stage, and activation of the embryonic genome. The induction of a full complement of sperm-induced egg activation events by ACh in the absence of sperm strongly suggests that sperm-induced egg activation is mediated by egg G proteins.

Acetylcholine↗
PubMed2005

Expression and possible involvement of calpain isoforms in mammalian egg activation.

At fertilization in mammals, the spermatozoon triggers a unique signal transduction mechanism within the egg, leading to its activation. It is well accepted that the earliest event observed in all activated eggs is an abrupt rise in intracellular calcium concentrations. However, little is known regarding the downstream proteins that are activated by this rise in calcium. Calpains constitute a family of intracellular calcium-dependent cysteine proteases whose members are expressed widely in a variety of cells. We investigated the expression and possible role of the calpain isoforms mu and m throughout egg activation. Both calpains were expressed in the rat egg and localized at the egg cortex as well as in the meiotic spindle. m Calpain translocated to the membrane and to the spindle area during parthenogenetic egg activation and during in vivo fertilization, upon sperm binding to the egg. The cytoskeletal protein alpha-spectrin (fodrin) was proteolysed by calpain during the egg-activation process, as demonstrated by specific calpain-breakdown products. Following parthenogenetic activation by ionomycin or puromycin, the calpain-selective permeable inhibitor, calpeptin, inhibited the resumption of meiosis and cortical reaction in a dose-dependent manner. Calpeptin was also effective in inhibiting in vitro fertilization. These results may imply a correlation between calpain activation and mammalian egg activation at fertilization and a possible role for calpain in the cascade of cellular events leading to resumption of meiosis.

Animals↗
PubMedDate not supplied

Protein tyrosine phosphatases in Chaetopterus egg activation.

Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTPsigma, -rho, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with beta-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately 140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process.

Acetophenones↗
PubMed2002

Signalling in mammalian egg activation: role of protein kinases.

Embryonic development is initiated after the fertilizing spermatozoon enters the egg and triggers a process known as 'egg activation'. Activation results in an increase in intracellular calcium concentration, cortical granule exocytosis (CGE), cell cycle resumption and recruitment of maternal mRNA. Various treatments can induce parthenogenetic activation characterized by the same manifestations. Signal transduction pathways similar to those known for somatic cells mediate the mammalian egg activation. This review focuses on the signal transduction pathways that occur during mammalian fertilization and during parthenogenetic egg activation. We discuss the possibility that members of the protein tyrosine kinase (PTKs) families, the Src family PTKs in particular, operate during egg activation and that protein kinase C can induce CGE.

Animals↗
PubMed1996

Intracellular pH change does not accompany egg activation in the mouse.

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pHi). We measured pHi in germinal vesicle (GV)-intact mouse oocytes, ovulated eggs, and in vivo fertilized zygotes using the pH indicator dye, SNARF-1. The mean pH, was 6.96 +/- 0.004 (+/- SEM) in GV-intact oocytes, 7.00 +/- 0.01 in ovulated, unfertilized eggs, and 7.02 +/- 0.01 in fertilized zygotes, indicating no sustained changes in pHi after germinal vesicle breakdown (GVBD) or fertilization. To examine whether transient changes in pHi occur shortly after egg activation, mouse eggs were parthenogenetically activated by 7% ethanol in phosphate buffered saline (PBS); no significant change in pHi followed ethanol activation. Since increased Na+/H+ antiporter activity is responsible for pHi increase in the sea urchin, pHi was measured in the absence of added bicarbonate or CO2 (a condition under which the antiporter would be the only major pHi regulatory mechanism able to operate, since the others were bicarbonate-dependent) in GV-intact oocytes, ovulated eggs, and in vivo fertilized zygotes to determine whether a Na+/H+ antiporter was activated. There was no physiologically significant difference in pHi after GVBD or fertilization, when pHi was measured in bicarbonate-free medium, nor any change upon parthenogenetic activation. Thus, a change in pHi is not a feature of egg activation in the mouse.

Animals↗
PubMed1995

Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs.

Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca2+ concentrations thought to be derived from inositol 1,4,5-trisphosphate-sensitive and -insensitive intracellular stores. Whereas the importance of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores has been evaluated, the identity and role of inositol 1,4,5-trisphosphate-insensitive stores are poorly understood. To explore the role of the ryanodine-sensitive Ca2+ store, we first used reverse transcription-polymerase chain reaction to identify transcripts of the ryanodine receptor in eggs and determined that transcripts for the type 2 and 3 receptor were present. Immunoprecipitation of radioiodinated egg extracts with an antibody that recognizes both type 2 and 3 receptors detected specifically a band of Mr = 520,000. Immunolocalization of the receptor(s) using laser-scanning confocal microscopy revealed that the receptor(s) was uniformly distributed in the cortex of the germinal vesicle-intact oocyte, but became asymmetrically localized to the cortex in a region apposed to the meiotic spindle in the metaphase II-arrested egg; this asymmetrical localization developed by metaphase I. The role of the ryanodine receptor in mouse egg activation was examined by determining the effects of microinjected ryanodine or cyclic ADP ribose on endpoints of egg activation in either uninseminated or inseminated eggs. Ryanodine induced the conversion of the zona pellucida glycoprotein ZP2 to its postfertilization form ZP2f in a biphasic concentration-dependent manner; nanomolar concentrations stimulated this conversion, whereas micromolar concentrations had no stimulatory effect. Cyclic ADP ribose also promoted the ZP2 conversion, but with a hyperbolic concentration dependence. Neither of these compounds induced cell cycle resumption. Inhibiting the inositol 1,4,5-trisphosphate-sensitive Ca2+ store did not inhibit the ryanodine-induced ZP2 conversion and, reciprocally, inhibiting the ryanodine-sensitive Ca2+ store did not inhibit the inositol 1,4,5-trisphosphate-induced ZP2 conversion. Last, treatment of eggs under conditions that would block the release of Ca2+ from the ryanodine-sensitive store had no effect on any event of egg activation following fertilization. Results of these experiments suggest that although ryanodine receptors are present and functional, release of Ca2+ from this store is not essential for sperm-induced egg activation.

Adenosine Diphosphate Ribose↗
PubMed1999

A 58-kDa Shc protein is present in Xenopus eggs and is phosphorylated on tyrosine residues upon egg activation.

A 58-kDa protein was detected in Xenopus egg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of the Xenopus Shc protein (p58 xShc) was found to increase 2.3 +/- 0.4-fold (n = 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RGDS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Calcium oscillations, sperm factors and egg activation at fertilisation.

When an egg is fertilised by sperm, the first intracellular signalling event observed is a large transient increase in cytoplasmic free Ca2+ ions. Elevated Ca2+ is known to play a vital role as an intracellular messenger in all cells and the Ca2+ signal occurring in the egg at fertilisation triggers the subsequent events that mediate early embryo development. In mammalian eggs, the Ca2+ response is first observed as a Ca2+ wave that initiates near the point of sperm-egg fusion, spreads across the entire egg, and then continues as a series of intracellular Ca2+ oscillations. The way in which the fertilising sperm generates the Ca2+ response in the egg has been the subject of much debate over recent years. One proposal for which there is growing evidence suggests the mechanism of egg activation at fertilisation involves the introduction of a soluble sperm protein into the egg shortly after sperm-egg fusion.

Animals↗
PubMed1993

Protein kinase C, a pivotal regulator of hamster egg activation, functions after elevation of intracellular free calcium.

We investigated the signal transduction pathways that mediate activation of Syrian hamster eggs. Under conditions in which the concentration of intracellular free calcium ([Ca2+]i) is clamped low, activation of protein kinase C (PKC) can induce second polar body formation, reformation of the nuclear envelope, and decondensation of chromatin, as well as golgi reformation. However, calcium is necessary for normal transition from meiotic metaphase II to anaphase II. Conversely, under conditions in which the level of PKC activity is clamped low, induction of a rise in [Ca2+]i, using the calcium ionophore A23187, does not induce egg activation. These results strongly suggest that PKC acts after the calcium signal as a proximal inducer of egg activation. This suggestion is supported by the kinetics of egg activation; PKC stimulators activate the eggs at a significantly enhanced rate (P < 0.01) compared with activation by calcium ionophore. We show here that PKC stimulators induce emission of the second polar body, but that subsequently, with longer culture, the emitted polar body is absorbed. Our results suggest that the rise in [Ca2+]i serves two functions, to activate PKC and to induce the transition from metaphase II to anaphase II. PKC, once activated, mediates several other events of egg activation.

Animals↗
PubMed2006

Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations.

Fertilization in mammalian eggs is accompanied by oscillatory changes in intracellular Ca(2+) concentration, which are critical for initiating and completing egg activation events and the developmental program. Ca(2+)/Camodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is postulated to be the downstream transducer of the Ca(2+) signal in many cell types. We tested the hypothesis that CaMKII is the major integrator of Ca(2+)-induced egg activation events and embryo development by microinjecting a cRNA that encodes a constitutively active (Ca(2+)-independent) mutant form of CaMKII (CA-CaMKII) into mouse eggs. Expression of this cRNA, which does not increase intracellular Ca(2+), induced a sustained rise in CaMKII activity and triggered egg activation events, including cell cycle resumption, and degradation and recruitment of maternal mRNAs; cortical granule exocytosis, however, did not occur normally. Furthermore, when mouse eggs were injected with sperm devoid of Ca(2+)-releasing activity and activated with either CA-CaMKII cRNA or by SrCl(2), similar rates and incidence of development to the blastocyst stage were observed. These results strongly suggest that CaMKII is a major integrator of the Ca(2+) changes that occur following fertilization.

Animals↗
PubMed1983

Increased free Ca2+ levels delay the onset of mitosis in fertilized and artificially activated eggs of the sea urchin.

Increasing free Ca2+ in the cytoplasm of fertilized and NH4OH- or A23187-activated eggs of Strongylocentrotus purpuratus and Lytechinus variegatus resulted in significant delays in the onset of mitosis. The increased Ca2+ levels were induced by addition of the calcium ionophore A23187 to suspensions of fertilized and activated eggs, or by fertilizing NH4OH-activated eggs. The delays may represent a temporary inhibition of the activation and activities of the mitotic proteins. It is postulated that relatively high free Ca2+ levels in the cytoplasm prevents activation of the mitotic proteins. Active Ca2+ sequestration is therefore suggested as an essential factor in the onset and progression of early mitosis.

Ammonium Hydroxide↗
PubMed1997

Evidence for an erbstatin-sensitive tyrosine kinase functioning in ascidian egg activation.

It has been proposed that protein tyrosine phosphorylation plays an important role in sperm-induced egg activation. To obtain evidence for the involvement of tyrosine kinases at an early stage in the egg activation process, we analyzed the effects of tyrosine kinase inhibitors on surface contraction of fertilized eggs of the ascidian Ciona savignyi and analyzed their effects on cleavage. We found that only erbstatin analog inhibited surface contraction, which was observed 5 min after insemination. With respect to the cleavage that occurs around 50 min after insemination, tyrophostin A1 and genistein, together with erbstatin analog, showed inhibitory effects. In addition, transient tyrosine phosphorylation of at least five proteins was observed 2-5 min after insemination, followed by tyrosine phosphorylation of one protein 30-40 min after insemination. Among proteins tyrosine-phosphorylated at the former stage, tyrosine phosphorylation of a 75 kD protein was inhibited by erbstatin analog. Thus, an erbstatin-sensitive tyrosine kinase functions at an early stage in the ascidian egg activation process.

Animals↗
PubMed2003

Regulation of maternal transcript destabilization during egg activation in Drosophila.

In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation.

Animals↗
PubMed1995

Calcium influx, fertilisation potential and egg activation in Fucus serratus.

Fertilisation in the marine alga Fucus serratus is accompanied by increased influx of Ca2+ from the external medium. The onset of this increase, monitored with the Mn2+ fluorescence quench technique, corresponded with the depolarisation phase of the fertilisation potential. External Ca2+ was necessary for the onset of the fertilisation potential and the early activation events, including cell wall exocytosis. Removal of Ca2+ from, or addition of Sr2+ to, the external medium during the fertilisation potential reduced the magnitude of the depolarisation and prolonged its duration. While fertilisation potentials could not be elicited in the presence of 0.1 mM Ca2+, addition of Ba2+ in the presence of 0.1 mM Ca2+ allowed normal fertilisation potential and egg activation. Microinjection of ryanodine or cyclic guanosine 5' -monophosphate (cGMP) did not induce cytoplasmic Ca2+ elevation or egg activation. Inositol 1,4,5-triphosphate [Ins(1,4,5)P3] produced a transient elevation of cytoplasmic Ca2+, monitored using ratio photometry, but did not cause cell wall exocytosis except at the site of microinjection. The results demonstrate an essential role for Ca2+ influx during Fucus egg activation. The relative importance of influx and intracellular Ca2+ release in Fucus egg activation is discussed.

Calcium↗
PubMed2004

Ca2+ oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation.

Sperm-specific phospholipase C zeta (PLC zeta) is known to induce intracellular Ca(2+) oscillations and egg activation when expressed in mouse eggs by injection of RNA encoding PLC zeta. We investigated the expression level and spatial distribution of PLC zeta in the egg in real time and in relation to the initiation and termination of Ca(2+) oscillations by monitoring fluorescence of a yellow fluorescent protein 'Venus' fused with PLC zeta. Ca(2+) oscillations similar to those at fertilization were induced at 40-50 min after RNA injection, when expressed PLC zeta reached 10-40 x 10(-15) g in the egg. PLC zeta-Venus increased up to 3 h and attained a steady level at 4-5 h. Interestingly, PLC zeta-Venus is accumulated to the pronucleus (PN) formed at 5-6 h and continuously increased there. Ca(2+) oscillations stopped in most eggs before initiation of the accumulation. A variant of PLC zeta that lacks three EF hand domains was much less effective in induction of Ca(2+) oscillations and little accumulated in the pronucleus, indicating a critical role of those domains. The ability of the accumulation to the pronucleus qualifies PLC zeta for a strong candidate of the Ca(2+) oscillation-inducing sperm factor, which is introduced into the ooplasm upon sperm-egg fusion and concentrated to the pronucleus after inducing egg activation.

Animals↗
PubMed1988

G-proteins and egg activation.

G-proteins are present in eggs, and experiments in which GTP-gamma-S, GDP-beta-S, cholera toxin and pertussis toxin have been injected into eggs have indicated the involvement of G-proteins in egg activation at fertilization and in oocyte maturation. Eggs into which serotonin or muscarinic acetylcholine receptors have been introduced by mRNA injection produce fertilization-like responses when exposed to serotonin or acetylcholine; since these neurotransmitter receptors act by way of G-proteins, this observation further supports the conclusion that a G-protein is involved in the fertilization process.

Animals↗
PubMed1993

The role of calcium in mammalian oocyte maturation and egg activation.

The maturation of the immature oocyte and the fertilization of a mature egg are two absolute prerequisites for mammalian embryo development. There is increasing evidence in mammals that both oocyte maturation and egg activation at fertilization are controlled by changes in intracellular free Ca2+ levels. The role of Ca2+ changes at fertilization is clear in that they are both required and sufficient for egg activation. However, it is not established how the sperm causes Ca2+ changes in eggs at fertilization, nor how different patterns of Ca2+ change affect embryo development. The role of Ca2+ in triggering oocyte maturation is less clear, although preventing intracellular Ca2+ changes can inhibit meiotic maturation at specific stages. Studies on how Ca2+ regulates meiosis and fertilization in mammals may provide new insights into the causes of failed fertilization in human IVF procedures.

Animals↗
PubMed2005

Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development.

Phospholipase C-zeta (PLCzeta), a strong candidate of the egg-activating sperm factor, causes intracellular Ca2+ oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLCzeta. Changes in the localization of expressed PLCzeta were investigated by tagging with a fluorescent protein. PLCzeta began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLCzeta in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLCzeta was recognized in every embryo up to blastocyst. Thus, PLCzeta exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca2+ oscillations in early embryogenesis.

Active Transport, Cell Nucleus↗